Objective The IOC recommends periodic cardiovascular screening of athletes, but the adoption of these recommendations is unknown. The purpose of this investigation was to evaluate and compare cardiovascular screening practices of countries participating in the Rio 2016 Olympic Games. Methods A list of chief medical officers (CMOs) was compiled by the IOC during the 2016 Olympic Games. CMOs were requested to complete an online survey about cardiovascular screening of their countries' Olympic athletes. Comparisons of screening practices were made by categorising countries by continent, gross domestic product (GDP) per capita and size of athlete delegation. Results CMOs for 117/207 (56.5%) countries participating in the 2016 Olympic Games were identified. 94/117 countries (80.3%) completed the survey, representing 45.4% of all countries and 8805/11 358 (77.5%) of all 2016 Olympic athletes. Most of the countries surveyed (70.2%) perform annual cardiovascular screening. Among the survey respondents, all or most athletes from each country were screened at least once with the following components: personal history (86.2% of countries), family history (85.1%), physical examination (87.2%), resting ECG (74.5%), echocardiogram (31.9%) and stress test (30.8%). Athletes were more likely to be screened with ECG in countries with relatively larger athlete delegation (OR 2.05, 95% CI 1.10 to 3.80, p=0.023) and with higher GDP per capita (OR 1.69, 95% CI 1.11 to 2.57, p=0.014). Conclusion Most of the responding countries perform annual cardiovascular screening of Olympic athletes, but there are differences in the components used. Athletes from countries with larger athlete delegations and higher GDP per capita were more likely to be screened with ECG.